mRNA fragmentation for 100bp paired-end reads

[amdic2]

Hi everybody,

I am currently planning a mRNA-seq experiment in a non-model species with the first goal being to study multiple copies elements expressed in the transcriptome.

TruSeq RNA sample preparation v2 Guide from Illumina suggests a 8min fragmentation step, which should give you a distribution of RNA fragment between 120bp to 210bp, with a median around 155bp.

However, as I am planning paired-end 100bp reads, it seems to me that I will loose some sequencing efforts (middle of inserts sequences twice).

- Have anyone tried to optimize fragmentation time as suggested on p.110 of this guide?
- What kind of fragmentation time have you used for paired-end reads?

Thanks!

Anne-Marie

[pmiguel]

We use 4 minutes at 94 oC for RNA libs that we want to do PE sequencing on.

Unless the fragmentation time table has been updated in v2, it is very wrong for the 0 second fragmentation. We tried 1 second and our resulting cDNA had a median size > 1kb when we ran an Agilent High Sensitivity DNA chip on it whereas Table 12 of the TruSeq RNA prep manual said the median insert size would be 200 bp!

Here are 3 libraries made with Soybean RNA. Red used standard 8 minute frag time, blue 4 minutes and green was 1 second. (These are DNA final libraries, heat denatured and run on an RNA Agilient chip to avoid the multimer (bird nesting) issue.)



Your mileage may vary, of course...

--
Phillip

[amdic2]

Phillip,
Thank you for the complete answer!
So it seems to me like the main difference between 4min and 8 min is the range, and not the median insert size. Am I correct?
I might try something like 2 and 4 minutes then. 8 minutes definitely seems to long...
Anne-Marie

[pmiguel]

I think the left side of the size distribution is determined by the ampure size selection after PCR. So, yes, I think decreasing the 94 oC time has the effect of spreading the size distribution farther to the right.

I would advise checking an aliquot of the double stranded cDNA on a high sensitivity Agilient chip, if you are tinkering with conditions. That will give you an idea of what you are going into the ligation with.

--
Phillip

[H Ernest Liu]

Hi,amdic2 and pmiguel!
I'm a new user attemping to perform the RNA-seq.Could you please send me a PDF file of the TruSeq DNA Sample Preparation v2 Guide.My email is[email protected]
Thanks a lot and best wishes~~

[H Ernest Liu]

The initial letter of my email is p

[ziseputi]

Will fragmentation buffer of mRNA-seq cut 200nt of my sample into small pieces?

Hi, Everyone,

More than 60% of my RNA sample is about 30-200nt, and other are from 200nt-2000nt, I am wondering whether the fragmentation buffer of mRNA-seq will cut 200nt of my sample into small pieces? so I can not sequence this part of RNA, or the fragmentation is random, so I can still get the sequence information of them? what is the best strategy if I want to get all the information of the RNA, is it necessary if I sequence the first library without fragmentation and the second with fragmentation? or it is not necessary to do so as the 200nt RNA will still exist? Thanks.

[amdic2]

Dear ziseputi,
The fragmentation used with this kit is chemical and thus random. You can then expect your fragments <200nt to be partially lost.
Sequencing two libraries could be an option, providing that you first do a size selection using beads to sequence preferentially the smaller fragments.
I hope this helps,
Anne-Marie